1. Field of the Invention
The present invention relates to the control of pathogenic microorganisms, particularly pathogenic fungi and certain bacteria. The invention also relates to lipopeptides having such antipathogenic activity i.e. high antifungal particularly against the Aspergillus genus and antibacterial activity. The invention also relates to the plants which have been treated with the lipopeptides or the microorganisms that synthesize and produce the lipopeptides. The invention also relates to other aspects further described herein.
An important objective of the invention is to address and to contribute to solve the aflatoxin problem that is caused by the aflatoxin-producing fungi, Aspergillus flavus and Aspergillus parasiticus.
2. Description of the Related Art
Bacillus ssp is known to produce a variety of peptide antibiotics that are antibacterial and/or antifungal. Although the peptides antibiotics are composed of amino acids, they often differed from gene-encoded polypeptides in their structure and mechanism of biosynthesis. Some are gene-encoded and synthesized ribosomally, but these often undergo posttranslational processing and modifications. Antibiotics produced non ribosomally are composed of 2 to 20 amino acids organized in a linear, cyclic or branched cyclic structure. Bacillus subtilis produced gene-encoded antibiotics and a variety of small antibiotic peptides with a molecular weight less than 2000 daltons, synthesized non-ribosomally. Subtilin is one gene-encoded lantibiotic peptide synthesized by B. subtilis as a prepropeptide that undergoes posttranslational processing (1). Among the antibiotics synthesized non ribosomally are two family: the lipopeptides including iturin, surfactin, fengycin, plistatin and the small hydrophilic di- and tripeptides. Iturin is a group of cyclic lipopeptides produced by Bacillus subtilis including iturin A, C, D and E (2,3), bacillomycin D, F and L (4), Bacillopeptin (5) and mycosubtlin (6). All contains a .beta.-amino fatty acid linked by amide bonds to the constituent amino acid residues of the iturin group. Iturin lipopeptide share a common sequence [.beta.-hydroxy fatty acid-Asx-Tyr-Asx] and show variation at the other four positions. Surfactin is also a cyclic lipopeptide containing seven residues of D- and L-amino acids and one residue of a .beta.-hydroxy fatty acid (7) with an amino acid sequence completely different from the iturin group. It is a powerful surfactant and has been described as an antifungal agent. Fengycin (8) and plipastatin (9) are lipopeptide with ten amino acid and a lipid attached to the N-terminal end of the molecule. They differed from iturin and surfactin by the presence of unusual amino acid such as ornithine and allo-threonine.
Table 1 shows the amino acid residues of the iturins and the chemical structure of Bacillomycin D.
TABLE I Amino acid residues Antibiotic L D D L L D L Major .beta. amino acid Iturin A Asn Tyr Asn Gln Pro Asn Ser n-C.sub.14, i-C.sub.15, a-C.sub.15 Iturin C Asp Tyr Asn Gln Pro Asn Ser n-C.sub.14, i-C.sub.15, a-C.sub.15 Bacillopeptin Asn Tyr Asn Ser Glu Ser Thr n-C.sub.14, i-C.sub.15, i-C.sub.16 Bacillomycin D Asn Tyr Asn Pro Glu Ser Tbr n-C.sub.14, i-C.sub.15, a-C.sub.15 Bacillomycin F Asn Tyr Asn Gln Pro Asn Thr i-C.sub.16, i-C.sub.17, a-C.sub.17 Bacillomycin L Asp Tyr Asn Ser Gln Ser Thr n-C.sub.14, i-C.sub.15, a-C.sub.15 Mycosubtilin Asn Tyr Asn Gln Pro Ser Asn i-C.sub.16, a-C.sub.17 Primary structure of iturins. Bacillomycin D ##STR1##
Iturin exhibit a restricted antibacterial activity and a broad range of antifungal activity against fungi and yeast but none of them has been shown to have antifungal activity against A. flavus although iturin A has already been patented for control of aflatoxin (10). Culture filtrate from B. subtilis were described in 1948 as antifungal against important dermatophytes and systemic fungi, and the undescribed antibiotic was named "Bacillomycin" (11). Since then 3 class of bacillomycin D (12), F (13) and L (14), have been characterized according to their amino acid sequence. Among each class, different antibiotics have been reported such as bacillomycin Fa, Fb, Fc, and Lc. Bacillomycin Fb and Fc differ from bacillomycin Fa by the presence of one or two carboxyl group respectively instead of carboxamide groups and bacillomycin Lc from bacillomycin L only by the sequence positions of a side chain amide and a carboxylic acid (15). An exhaustive study of the literature show that bacillomycin F has been tested against a wide range of fungi and bacteria (Table II).
TABLE II MIC (ug/ml) Bacillomycin Bacillomycin Bacillomycin Test Organisms Fb Fc Fa Aspergillus niger 25 30 40 Botrytis cinerea 20 Cladosporium cladosporioides 25 40 Fusarium oxysporum &gt;200 &gt;200 &gt;320 Mycosphaerella pinodes 100 30 10 Neurospora crassa 80 Penicillium chysogenum 20 Pleospora herbarum 10 Rhodotorula pillimanae 80 Sclerotina fructigena 40 Sclerotina sclerotiorum 50 Stemphylium radicinum 320 Trichophyton mentagrophytes 20 Candida albicans &gt;200 &gt;200 40 Candida tropicalis &gt;200 &gt;200 40 Saccharomyces cerevisiae 25 30 10 Azotobacter vinelandii &gt;400 Brucella brochiseptica &gt;400 Escherichia coli K12 &gt;400 Streptomyces albus G &gt;400 Bacillus cereus &gt;400 Micrococcus luteus 200 Sarcinia lutea &gt;400 Staphylococcus aureus &gt;400 Kluyveromyces bulgaris 75 40
Bacillomycin F showed a strong antifungal activity against various yeasts, fungi and phytopathogenic fungi but a weak antibacterial activity (16). Bacillomycin Lc has been described as a new antibiotic of the bacillomycin family with antifungal activity against phytopathogenic fungi such as Ophiostoma ulmi, Verticillium dahliae, Ceratocystis fagacearum and Cryphonectria parasitica the causal agents of Dutch elm disease, Verticillium wilt of maples, oak wilt and chestnut blight respectively. Bacillomycin D has been reported to be antifungal against Absydia corymbifera, Aspergillus Niger, Candida Albicans, Fusarium oxysporum, Kluyveromyces bulgaris and Saccharomyces cerevisae (17).
Mycosubtlin, iturin A and bacillomycin L inhibited the growth of Micrococcus luteus, their activity are different upon Micrococcus luteus protoplast. Mycosubtlin and Iturin A are able to lyse Micrococcus luteus protoplast and bacillomycin L has no effect (18). Even a slight modification of the molecule as the methylation of the aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity (19). The phenolic group of the tyrosine residue has been shown to be essential for the antifungal activity, when the tyrosine residue was substituted no activity was observed (20).
Aflatoxin Problem
The invention relates and contributes to solve an important scientific problem of serious economic and business consequences in the United States and in the world. To date, there is no acceptable and effective way and means to control aflatoxin on plants susceptible to aflatoxin-producing fungi. There are no known aflatoxin-resistant plants, genetically transformed or otherwise.
Drought stress and high temperatures at critical times during kernel or seed development and/or insect injury of crops can contribute heavily to aflatoxin contamination of corn by the aflatoxin-producing fungi, A. flavus and A. parasiticus. The aflatoxin-producing fungi, A. flavus and A. parasiticus, present health hazards to humans and animals through the toxic and carcinogenic properties of their secondary products. Even a very low level of aflatoxin contamination can lead to severe economic losses in the peanut industry. To control this problem, breeding peanut varieties resistant to Aspergillus sp. or which support less aflatoxin production has been attempted with limited success.
No commercial corn hybrids with high resistance to either infection by A. flavus or to aflatoxin accumulation are currently available. Aflatoxin detected at levels of 20 parts per billion (ppb) (established by the FDA) or above can make the crop unsalable. Aflatoxin is considered to be toxic to livestock being fed contaminated grain and to be a carcinogen, correlated with liver cancer, in certain human populations around the world. Cottonseed grown in the Yuma Valley, Ariz., corn grown in certain states of the southern U.S. and peanuts grown in certain regions of Georgia are chronically contaminated with aflatoxins resulting in direct losses by farmers amounting to tens of millions of dollars annually. Corn grown in the mid western states are contaminated with aflatoxin on a sporadic basis. Exact economic losses due to sporadic or limited aflatoxin outbreaks are difficult to determine, particularly in terms of detrimental impact at each of the various steps in the marketplace (including foreign markets). However, during drought years (1983 and 1988) in the mid west corn belt, which led to large aflatoxin outbreaks, losses were estimated to total in the hundreds of millions of dollars.
A major outbreak of aflatoxin in Southern Grown Corn, occurred recently in 1998. Aflatoxin contamination of corn in Arkansas, Georgia, Louisiana, Mississippi, Missouri, South Carolina, Tennessee, and Texas in 1998 was one of the worst ever recorded. A potentially record number of grain-load rejections, due to aflatoxin levels in excess of FDA limits, occurred in some southern states. The aflatoxin outbreak in 1998 has additional significance since corn production in certain southern states has probably tripled in the past three years. It is certainly no coincidence that continuous days of 90(+) F temperatures and low or no rainfall dominated weather conditions for significant portions of the 1998 growing season in Southern states. The literature indicates that these conditions are highly conducive, even necessary for large aflatoxin outbreaks in susceptible crops. Insect injury is often suggested to be the major mode of entry by aflatoxin producing fungi. However, insect injury apparently does not account for the enormity of the aflatoxin outbreak experienced this past year in the South. Thus, controlling insects (e.g. cultivation of transgenic corn containing Bt, an insect toxin), although an essential part of good management practices, would not necessarily have been effective in reducing aflatoxin levels in Southern-grown corn in 1998. Some observations suggest that the fungus gained entry by some other, little understood means associated with high temperature and drought conditions. This highlights the need for extensive research on drought and temperature effects on plant health and physiology and fungal virulence and to find a solution to these problems.
In accordance with the invention, a method, microorganisms of the Bacillus family, peptides synthesized by the Bacillus, especially by B. subtilis have been discovered that contribute an important technical advance to the resolution of the aflatoxin problem.